AlignmentViewer

Basically, you need not particular operation after having downloaded the program file (AlignmentViewer.jar) — double click the icon (OSX or Windows) or type the java command (Unix) to invoke the program. See the chapter " The CGAT AlignmentViewer program "

However, in the default setting of the Java virtual machine (JVM), you will be likely to encounter a memory allocation error especially when aligning long sequences dynamically. To avoid this problem, you need to increase the size of the memory allocation pool for the JVM.

• Windows: Download the AlignmentViewer.jar file and do the following (see Figure 1).

  1. Create a shortcut of the AlignmentViewer.jar file (Right-click on the jar file ⇒ Create Shortcut).

  2. Open the properties window of the shortcut (Right-click on the shortcut ⇒ Properties).

  3. Open the Shortcut tab and enter the java command in the Target: box as follows: [ java -Xmx512m -jar "C:\Documents and Settings\..." ], where "C:\Documents and Settings\..." is the original file location. This might be different in you situation, but do not modify the original name in anyway. Then, press "OK".

• Mac OSX: Download and use the OSX application version of AlignmentViewer (AlignmentViewer.app), instead of the AlignmentViewer.jar file.

• Linux/Unix: Download the AlignmentViewer.jar file and type java -Xmx512m -jar AlignmentViewer.jar to invoke the program.

DataServer

Copy the downloaded archive CGAT.tgz into an appropriate directory and extract files by tar command, which generates a directory named CGAT. Enter this directory and invoke a configuration script SetUp.pl.

tar cvfz CGAT.tgz
cd CGAT
SetUp.pl

# in csh or tcsh
source etc/cgat.csh

# in sh or bash
. etc/cgat.sh

The set up script tries to detect alignment programs available on the server machine and installs detected programs in the $CGAT_HOME/build/align directory. You might want to move some commands from the $CGAT_HOME/build/align.in directory or to remove some unnecessary commands manually.

Similarly, you might want to install manually some optional commands for collecting feature segments from the $CGAT_HOME/build/segment.opt directory. Note that in this case you must always set up by yourself (if you wanted), since currently the set up script does not try to check the availability of these commands automatically. Currently, the following commands are available:

• Glimmer: Microbial gene-finding system available at http://www.cbcb.umd.edu/software/glimmer/. Currently, ver.2 is assumed by default, but you can use ver.3 if you manually edit the commands/Glimmer.pl file. Before that, of course, you must correctly set up the glimmer program itself.

• ISfind: Searching an insertion sequence (IS) database by the BLAST program. The IS database should be a FASTA formatted sequence file with each comment line containing IS family name as ">${ISname} Fam:${familyname}", and the file should be installed as $CGAT_HOME/database/isdb . See the IS Finder database for information about IS families. One can use the GIB-IS database available at DDBJ as a downloadable IS database. Download the table file (IS-table-text.txt) and run the following command:

  $CGAT_HOME/build/bin/conv_GIB_IS.pl -nr IS-table-text.txt > $CGAT_HOME/database/isdb

Load data

After running the program, first you must load data by choosing File ⇒ Load from the menu.

Control panel

The control panel contains several buttons for controlling the current view of both the alignment and dotplot displays.

Alignment display panel

The alignment display panel contains three basic tracks: the central tracks (alignment track) shows a graphical representation of the alignments, and the upper and lower tracks (gene annotation tracks) show the gene annotation of the upper and lower genomes, respectively. More annotation tracks can be added to display the location of several feature segments.

In addition, the "location fields" and the "strand switches" are on the left side of the tracks. The location field indicates the central coordinate of the current view, and strand switch indicates the strand of each genome currently selected. Users can jump to the desired location by entering a coordinate value or a gene identifier (e.g. HP0812) in the location field, and can toggle the strand direction by pressing the strand switch.

Some notes: 1) You must always enter the location of the reference genome if you want to see the orthologous alignment around the specified locus. If you want to specify the location in the current target genome, you should exchanges the reference and the target genomes before entering the position; 2) If you invert the reference genome by the strand switch button, the direction of the subsequent movement is also inverted. This means that when you press the 'move left' button, the cursor in the dotplot display will move right, since the coordinate of the dotplot display is not changed by this operation.

Dotplot display panel

In the dotplot display panel, all alignments are plotted onto the 2-dimensional space with the horizontal (X) and the vertical (Y) axes representing the reference and the target sequences, respectively. In this plot, the current scope, i.e. the region displayed in the alignment display, is also shown as a rectangular region. Users can move the current scope by clicking on any points in the plot to move.

In the dotplot display, independent scaling of the x-axis and y-axis is possible. This feature is useful in visualizing the distribution of homologous regions of a specific segment on one genome against the entirety of the other genome. In the following example, dotplot with expanded y-axis scale clarifies the presence of inverted duplication at both ends of the rearranged segment on the reference genome (x-axis).

File menu

Search menu

View menu

Directory structure

Overview of the data construction procedure

Essentially, the data construction process consists of the following steps:

1. Prepare your genomic sequence data. If your sequences are already in the MBGD database, you can skip this step, because the procedure will automatically try to download missing data from MBGD.

2. Edit $CGAT_HOME/etc/speclist to specify a set of species to be analyzed and a set of programs to be executed for each species set.

3. Run $CGAT_HOME/build/BuildAll.pl. This script executes all procedures for building the database according to the $CGAT_HOME/etc/speclist file, and eventually constructs data on the $CGAT_HOME/database.work directory.

4. Run $CGAT_HOME/build/Release.pl to release the data from the $CGAT_HOME/database.work directory to the $CGAT_HOME/database directory.

Preparing genomic sequences

Before running programs, you must prepare genome sequences data. There are three possible ways.

1. If your sequences are already in the MBGD database, the simplest way to prepare data is to remember the abbreviated names of those genomes in the MBGD database (e.g. 'eco' for Escherichi coli K12). and use these names in the configuration table described in the next section. The build procedure automatically try to download missing data from MBGD. Note that the sequence name specified here is used throughout the system (hereafter we call it SPNAME).

2. Alternatively, if you have genomic data in GenBank format, you can use the $CGAT_HOME/build/getDataFromGenBank.pl script to convert it to the CGAT database. The syntax of the command is

   getDataFromGenBank.pl GBK_FILE SPNAME

   For example:

   getDataFromGenBank.pl NC00913.gbk eco

   Here, you can use as SPNAME any name that is composed of alphanumeric characters, provided that it is unique.

3. Otherwise, you must prepare data by yourself. The data you should prepare are as follows:

   • Choose an appropriate unique name (SPNAME) that is composed of alphanumeric characters.

   • $CGAT_HOME/database/genomes/SPNAME: a genomics sequence in fasta format.

   • $CGAT_HOME/database/genes/aa/SPNAME: translated sequences of genes in fasta format (optional; if you want to calculate attribute values associated with protein sequences).

   • $CGAT_HOME/database/genes/nt/SPNAME: nucleotide sequences of genes in fasta format (optional; if you want to calculate attribute values associated with nucleotide sequences).

   • $CGAT_HOME/database/genes/tab/SPNAME: a tab-delimited table of genes containing the following information: beginning position, ending position, direction (1/-1), color code (= function category code; optional), the name of the gene, and the name of the product (optional). The beginning position must be smaller than the ending position even for the gene on the reverse chain. The file must begin with a header line containing a tab-delimited list of field names as follows,

     #from to dir color name product 190 255 1 1 B0001 thr operon leader peptide 337 2799 1 1 B0002 bifunctional aspartokinase I/homeserine dehydrogenase I 2801 3733 1 1 B0003 homoserine kinase 3734 5020 1 1 B0004 threonine synthase 5234 5530 1 100 B0005 hypothetical protein

     By default color code is defined base on the MBGD function category. Specify '100' if you do not need to assign any color. You can change the color code by modifying $CGAT_HOME/etc/colorTab/colorTab.gene.

Configuring database building procedure

Next, you must prepare $CGAT_HOME/etc/speclist file, which contains information required for the database building procedure. The following information should be specified in this file:

• A set of genomic sequences to be compared. If more than two species are specified, all-against-all pairwise comparison will be performed. Note that currently CGAT can handle only pairwise comparisons.

• A set of programs to be executed for each sequence set.

The speclist file consists of macro definition and dataset definition sections, where macro definition must precede the dataset definition. Syntax of the definition of macro variable is as follows:

SET varname = value

The dataset definition section is a tab-delimited table, containing the following fields: SPNAME_LIST, PROGRAM_LIST,FLAG_UPDATE, and FLAG_PUBLIC. SPNAME_LIST is a comma-delimited list specifying a set of species to be compared. PROGRAM_LIST is a space-delimited list specifying script files to be executed for each species set. The file names are relative to the $CGAT_HOME/build directory and the wildcard characters such as '*' can be used. For example, align/* specifies all (executable) files under the $CGAT_HOME/build/align directory. FLAG_UPDATE and FLAG_PUBLIC are flags specifying the data should be updated and the data should be open to the public (through the CGI script), respectively. The value should be 1 (yes) or 0 (no). Default value is 1.

The following is an example of the speclist file. The file directs the build script to compare genome sequences "hpy" and "hpj" by executing all programs for identifying feature segments in the $CGAT_HOME/build/segment directory, followed by all programs for calculating gene attribute values in the $CGAT_HOME/build/geneattr directory, followed by all programs for calculating alignment between the two genomes n the $CGAT_HOME/build/align directory.

####################
# macro definition
####################
SET AlignAll = align/*
SET SegmentAll = segment/* geneattr/*
####################
# dataset definition
####################
hpy,hpj	$SegmentAll $AlignAll

Start building

Run BuildAll.pl under the $CGAT_HOME/build directory to execute building procedures. This script creates $CGAT_HOME/work directory if not exists, and move to this directory, and then executes programs that have been specified in the $CGAT_HOME/etc/speclist file in the previous section.

BuildAll.pl

Release data

Before accessing the created database, you must run Release.pl under the $CGAT_HOME/build directory to release the data from the $CGAT_HOME/database.work directory to the $CGAT_HOME/database directory.

Release.pl